Carbon dioxide is chemically stable and unreactive, and must be reduced to enable its incorporation into biological molecules. Autotrophic microorganisms are able to utilize carbon dioxide as their sole carbon source and a variety of pathways are known to activate and incorporate it into biomolecules essential for growth and replication. Recently, carbon dioxide fixation pathways have received interest for biotechnological applications, since this could provide biological routes for de novo generation of fuels and small organic molecules (Hawkins et al., 2011, ACS Catal. 1, 1043-1050).
There are currently at least six natural pathways for the incorporation of inorganic carbon dioxide into cellular carbon (Berg 2011, Appl. Environ. Microbiol. 77, 1925-1936; Berg et al., 2010, Nat. Rev. Microbiol. 8, 447-460). The most recently discovered of these are found exclusively in extremely thermophilic archaea: the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle, which operates in members of the crenarchaeal order Sulfolobales ((Berg 2011, Appl. Environ. Microbiol. 77, 1925-1936; Berg et al., 2007, Science 318, 1782-1786; Alber et al., 2008, J. Bacteriol. 190, 1383-1389; Hugler et al., (2003) Arch. Microbiol. 179, 160-173), and the dicarboxylate/4-hydroxybutyrate (DC/4HB) cycle, which is used by anaerobic members of the orders Thermoproteales and Desulfurococcales (Berg et al., 2007, Science 318, 1782-1786; Huber et al., (2008) PNAS USA 105, 7851-7856). In both cycles, two carbon dioxide molecules are added to acetyl-CoA (C2) to produce succinyl-CoA (C4), which is subsequently rearranged to acetoacetyl-CoA and cleaved into two molecules of acetyl-CoA. These pathways differ primarily in regards to their tolerance to oxygen and the co-factors used for reducing equivalents—NAD(P)H for the 3HP/4HB cycle and ferredoxin/NAD(P)H for the DC/4HB cycle (Berg et al., 2010, Nat. Rev. Microbiol. 8, 447-460; Auernik and Kelly, 2010, Appl. Environ. Microbiol. 76, 931-935). The two archaeal pathways also differ in how they link the CO2 fixation cycle to central metabolism. In the DC/4HB pathway, pyruvate is synthesized directly from acetyl-CoA using pyruvate synthase. In the 3HP/4HB pathway, another half-turn is required to make succinyl-CoA, which is then oxidized via succinate to pyruvate (Berg 2011, Appl. Environ. Microbiol. 77, 1925-1936; Ramos-Vera et. al., 2011, J. Bacteriol. 193, 1201-1211; Estelmann et al., (2011) J. Bacteriol. 193, 1191-1200).
There are 13 enzymes proposed to catalyze the 16 reactions in the 3HP/4HB pathway. The first three enzymes convert acetyl-CoA (C2) to 3HP (C3) via an ATP-dependent carboxylation step. Next, 3HP is converted and reduced to propionyl-CoA, carboxylated a second time and rearranged to make succinyl-CoA (C4). Succinyl-CoA is reduced to 4HB, which is converted to two molecules of acetyl-CoA in the final reactions of the cycle. Flux analysis and labeling studies have confirmed the operation of this pathway in M. sedula (Berg et al., 2007, Science 318, 1782-1786; Estelmann et al., (2011) J. Bacteriol. 193, 1191-1200).